Many neurotropic flaviviruses can invade the central nervous system (CNS), infect resident cells and establish acute or persistent infections. Thus, successful attenuation of viral neuropathogenesis depends on prevention of virus entry into the CNS and restriction of its replication in the neurons. To limit virus access into CNS, attenuated vaccine candidates against neurotropic JEV, WNV, SLEV and TBEV are being developed in the NFS using the strategies based on chimerization of a neurovirulent virus with a non-neuroinvasive dengue type 4 flavivirus (DEN4) or naturally attenuated tick-borne Langat virus (LGT) and introduction of attenuating mutations. To restrict viral replication in the neurons, we have developed and utilized an effective strategy for selective control of virus neurotropism by targeting of viral genomes for cellular microRNAs (miRNAs) expressed in the brain. The most attenuated vaccine candidates were then evaluated for safety, immunogenicity, and their ability to protect mice and monkeys against challenge with wild-type virulent virus. In addition, a miRNA targeting approach was adapted to design environmentally-safe vaccine viruses restricted in their ability to infect and be transmitted by competent arthropod vectors. JEV, SLEV and WNV vaccines: Our WNV vaccine is a chimeric virus in which the structural prM and E protein genes of the DEN4 virus have been replaced by those of wild-type (wt) WNV. In preclinical studies, a vaccine virus was unable to establish reproductive or persistent infection in the CNS of non-human primates - infectious virus and viral antigens were not detected in the brain or spinal cord. In clinical trials (Pierce et al., J Infect Dis 2017), the WNV vaccine was found to be safe, well-tolerated, and effective in healthy elderly volunteers, adults aged 50-65 years. Immunization with a single vaccine dose produced significant level of neutralizing antibody (NTAb) against wt WNV strains, inducing a 95% rate of seroconversion in the volunteers. These results suggest that a single dose of vaccine may be sufficient to induce protective immunity in the vulnerable elderly population. In FY2018, the new generation of vaccine candidates was engineered by introducing multiple targets for the CNS-specific miRNAs into WN/DEN4, JE/DEN4 and SLE/DEN4 genomes. Each monovalent vaccine candidate (JE/DEN4mirT, WN/DEN4mirT or SLE/DEN4mirT virus) demonstrated reduced neuroinvasiveness in immunocompromised SCID and B6 IFNRI-KO mice, which lack the type I interferon receptor. In immunocompetent animals, each monovalent vaccine candidate provided complete protection against homologous JEV, WNV or SLEV lethal challenge, but only partial cross-protection was observed against heterologous challenge, indicating that immunity to one virus (JEV, WNV or SLEV) would not be sufficient to protect against another member of the JEV serocomplex. However, a single dose of bivalent vaccine formulations (admixture of miRNA-targeted viruses: WN/DEN4 + SLE/DEN4, JE/DEN4 + WN/DEN4, or SLE/DEN4 + JEV/DEN4) induced strong NTAb responses and protected mice against lethal challenge with parental viruses. Currently, we are working to identify a dose of each component in the different admixtures of combined trivalent vaccine to achieve the balanced antibody response and protection against challenge with virulent wt JEV, SLEV and WNV viruses. TBEV vaccines: The TBEV vaccine candidates are chimeric viruses carrying the prM and E structural protein genes of Far Eastern TBEV and remaining sequences derived from either (1) a non-neuroinvasive DEN4 (TBEV/DEN4), (2) a naturally attenuated, tick-borne LGT strain E5 (TBEV/E5) or (3) a most immunogenic LGT strain T1674 (TBEV/LGT). A large set of modified TBEV/DEN4 and TBEV/LGT was generated by introducing the cassettes of brain-specific miRNA-targets into three distant regions of chimeric DEN4- or LGT-based genomes in the duplicated capsid gene region, the duplicated E gene region, and the 3'NCR. In FY2018, we compared the most promising TBEV/DEN4mirT, TBEV/LGT (E5)mirT and TBEV/LGT(T1674)mirT vaccine candidates in brain-infected mice for severity of virus-associated histopathology in the CNS and for assessment of the responses of the CNS resident cells (microglial activation and neuronal degeneration) as well as for virus genetic stability. We found that the microRNA co-targeting in three distant regions of TBEV/DEN4mirT or TBEV/LGTmirT severely restricted virus replication in the brain, improved genetic stability, completely abolished virus neurotropism, and strongly reduced virus-induced neuropathogenesis. We have found that TBEV/DEN4mirT and TBEV/LGTmirT-based on the genetic background of LGT strain 1674 are more immunogenic in mice and rhesus monkeys as compared to TBEV/LGTmirT-based on LGT strain E5. In addition, the level of TBEV-specific NTAb induced by a single dose of these viruses in monkeys was comparable to that induced by three human doses of a licensed inactivated TBEV vaccine and was able to provide protection against severe TBEV challenge. In FY2018, using Langat virus as a model, we developed a dual strategy for virus attenuation which synergistically accesses the specificity of miRNA genome targeting and the effectiveness of internal ribosome entry site (IRES) insertion. In this novel approach (Tsetsarkin et al., mBio 2017), the capsid protein gene was relocated into the 3 noncoding region and expressed under translational control of an IRES. Engineered bicistronic LGTs carrying multiple target sequences for brain-specific miRNAs were stable in Vero cells and induced adaptive immunity in mice. Importantly, miRNA-targeted bicistronic LGTs were not pathogenic for either newborn mice after intracranial inoculation or adult immunocompromised mice (SCID or B6 IFNRI-KO) after intraperitoneal injection. Moreover, bicistronic LGTs were restricted for replication in tick-derived cells, suggesting an interruption of viral transmission in nature by arthropod vectors. This approach is suitable for reliable attenuation of many RNA viruses. ZIKV pathogenesis: Recently, we generated a full-length infectious cDNA clone of ZIKV isolated during 2015 epidemic in Brazil (Tsetsarkin et al., mBio 2016) and used it as a convenient genetic platform for studies of virus-host interactions and vaccine development. In FY2018, we created a panel of viruses expressing complementary targets for the CNS-specific cellular mir-9 and mir-124 and for miRNAs enriched in male and female reproductive organ tissues. We traced dissemination of these viruses in the male reproductive tract of AG129 mice deficient for type I and type II interferon receptor genes and compared them with virus dissemination into the CNS. We demonstrated that ZIKV infection of the testis and CNS can be restricted independently by inserting targets for miRNA selectively expressed in these organs. In contrast, ZIKV infection of epididymis can only be blocked by co-targeting of the virus for testis- and epididymis-specific miRNAs. This suggests that two routes of infection are available for ZIKV in the epididymis: (1) direct infection from the blood and (2) testicular infection with the flow of infected sperm. Finally, we showed that co-targeting the ZIKV genome for the combination of CNS-, testis- and epididymis-specific miRNAs restricts ZIKV infection of these organs but does not impair the development of virus-induced humoral immunity in mice. These defined alterations in ZIKV organ tropism are an important step in the development of miRNA targeted live-attenuated ZIKV vaccine.